1. Field of the Invention
The present invention relates to imidazolium salts, the respective carbene metal complexes as well as their utilisation in bioanalytical tags for biomolecules.
2. Related Technology
The research into the structure and functionality of biomolecules like proteins and carbohydrates as well as genomes and gene transcripts requires either special derivatives and/or mass spectrometric analysis. Methods used are for instance ESI (Electron Spray Ionization) [I], MALDI (Matrix-Assisted Laser Desorption Ionization) [2] and ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) [3-5]. Such processes cause separation and purification steps by means of liquid chromatography (LC) or high performance liquid chromatography (HPLC) [6]. This requires techniques that are a combination of purification and analysis and are usually named by an acronym that combines the names of the individual techniques (hyphenated techniques).
The structure and the sequential architecture of the named biomolecules make great demands on the quantitative analysis. Particularly, the absence of quantitatively measurable functional groups and the limited availability of suitable heteroatoms respectively complicate the application of analytic methods like fluorescence spectroscopy or UV-vis spectroscopy as well as the use of electroanalytical methods. Even ICP-MS becomes more difficult or impossible [6] without an additional derivatisation step. Often, a so-called derivatisation step occurs, whereby a chromophoric, fluorophoric or electroactive group is incorporated into the analyte. The introduction of a heteroelement as respective derivatisation step enables access to ICP-MS studies [7].
For various medicinal-chemical applications, initial derivatisation steps were developed for the routine determination of certain analytes and were introduced into the respective protocols. The greatest disadvantage of this is the limitation to the simultaneous detection of only a single analyte. For the detection of more than one analyte, multiple detection sequences are necessary. The simultaneous detection is presently impossible. Protocols for the simultaneous detection of more than one analyte, preferably by applying identical analytical methods, are therefore urgently needed [8].
The simultaneous measurement of different analytes by means of tandem mass tags (TMT) or isobaric data recognition (isobaric tagging) for relative and absolute quantification (iTRAQ) [9,10] is possible in the field of proteomics. But, isobaric tagging techniques require marking with heavy isotopes (2D, 13C, 15N) in an expensive, multistep synthesis, although the tags are commercially available.
Tagging methods should provide stable tags that allow a low detection limit and are non-toxic and inexpensive at the amounts used. Further, it should be possible to introduce them quantitatively and specifically. A broad range of individual tags should be available, which enables the simultaneous detection of different analytes.
Organometallic compounds or metal coordination compounds have the potential to fulfil all these demands. Several classes of compounds have already been synthesized and have found application as bioanalytical tags (BAT) [6]. In this sense “stable” means the compound is stable towards air and moisture and is usable for coordination compounds and for organometallic compounds. But “stable” also means stable with respect to leaching and in general towards displacement from the biomolecule. In the case of metal containing BATs, this means a strong coordination of the ligand(s) to the metal, which should be possible best by means of a combination of chelate ligands and N-heterocyclic carbenes (NHC) [11,12].
Promising classes of compounds that are usable for BAT applications are metal carbonyls, carbene complexes (Fischer and Wanzlick-Arduengo), as well as ferrocene and chelate complexes [6]. But, metal carbonyl complexes are susceptible to an early loss of carbonyl ligands in mass spectrometry, which complicates the analysis of spectra [13]. On the other hand, they offer a unique window of detection for IR-spectroscopy [14]. Ferrocenes are definitely the furthest developed class of compounds for electrochemical detection. But here, too, there are limitations with respect to derivatisation and thus multiple determination of analytes.
In total, not even ferrocenes can simultaneously combine the advantages of electrochemical detection, unique isotope patterns for MS-detection and a broad modification range.